Repurposing Endonuclease V for the detection and engineering of adenosine-to-inosine editing

项目总结/摘要 RNA编辑是细胞广泛采用的一种转录后改变蛋白质序列和基因的策略。 表达水平。腺苷到肌苷（A-to-I）编辑是这些RNA中最常见和最有影响力的一种 RNA修饰是通过腺苷脱氨酶（ADAR）催化的。脱氨作用改变了 核碱基的结构和氢键模式，并且所得的肌苷与胞嘧啶杂交， 有效地将这些位点重新编码为鸟嘌呤。编辑对许多过程至关重要，包括胚胎基因- 姐妹，神经功能和先天细胞免疫，功能失调编辑也与自身免疫有关 疾病、神经系统疾病和几种癌症。尽管A-to-I编辑在细胞内的关键作用， 功能，我们对这种修改的位置和频率的理解受到固有限制的限制， 在目前可用的用于映射和定量转录组中的肌苷的方法中，此外，本发明还 位点选择性诱导A-to-I编辑的能力对于这种修饰的研究都是非常有价值的， 阳离子和新的治疗方法的发展，但目前的方法受到他们的依赖 天然或修饰的阿达尔酶的底物结合偏好。一个核心挑战阻碍了 用于研究A-to-I编辑的方法的发展是缺乏抗肌苷抗体或其他抗肌苷抗体的可用性。 能够选择性结合该修饰的核苷酸的亲和试剂。我们克服了这一挑战， 将天然存在的EndoV蛋白从RNA切割酶再利用为RNA结合蛋白 并利用这一点开发了从总细胞RNA中富集含肌苷RNA的工作流程。我们有 表明这增加了RNA-seq数据中含有肌苷的读数的比例，并促进了发现 转录组中新的A到I编辑位点。拟议的研究将利用我们的EndoV方法， 开发一个技术工具箱，以推进A-to-I编辑的研究和工程。总之，这些新 这些方法将使研究人员能够更准确地绘制转录组中的编辑位点，量化转录组中的变化， 快速且高通量地进行总体编辑流行，以及在活细胞中的特定靶位点进行直接编辑。 此外，这里开发的方法可以应用于除了ino之外的其他表位转录组修饰。 正弦，提供了一套对RNA编辑社区具有广泛实用性的技术。

Project Summary/Abstract RNA editing is a widespread strategy employed by cells to post-transcriptionally alter protein sequence and gene expression levels. Adenosine-to-inosine (A-to-I) editing is among the most common and impactful of these RNA modifications and is catalyzed by adenosine deaminases acting on RNA (ADARs). Deamination changes the structure and hydrogen bonding pattern of the nucleobase, and resulting inosines hybridize with cytosine to effectively recode these sites as guanine. Editing is essential for a number of processes including embryogene- sis, neurological function, and innate cellular immunity, and dysfunctional editing is also linked to autoimmune diseases, neurological disorders, and several types of cancer. Despite the critical role of A-to-I editing in cellular function, our understanding of the locations and frequency of this modification are confined by the inherent limi- tations in the currently available methods for mapping and quantifying inosine in the transcriptome. Additionally, the ability to site-selectively induce A-to-I editing would be extremely valuable for both the study of this modifi- cation and the development of new therapeutic approaches, yet current methods are hampered by their reliance on the substrate binding preferences of native or modified ADAR enzymes. A central challenge that has hindered the development of methods for studying A-to-I editing is the lack of availability of anti-inosine antibodies or other affinity reagents capable of selective binding to this modified nucleotide. We have overcome this challenge by repurposing the naturally occurring EndoV protein from an RNA-cleaving enzyme into an RNA-binding protein and have used this to develop a workflow to enrich inosine-containing RNAs from total cellular RNA. We have shown that this increases the fraction of reads in RNA-seq data that contain inosine and facilitates the discovery of new A-to-I editing sites in the transcriptome. The proposed research will leverage our EndoV method to de- velop a toolbox of technologies to advance the study and engineering of A-to-I editing. Together, these new methods will enable researchers to more accurately map editing sites in the transcriptome, quantify changes in overall editing prevalence rapidly and in high-throughput, and direct editing at specific target sites in living cells. Additionally, the methods developed here can be applied to other epitranscriptomic modifications beyond ino- sine, providing a set of technologies that are of broad utility to the RNA editing community.